Retroviral vectors for delivery of interfering RNA

ABSTRACT

Provided herein are retroviral vectors for delivering interfering RNA into cells.

FIELD OF THE INVENTION

The present invention relates generally to retroviral vectors fordelivering interfering RNA into a cell.

BACKGROUND OF THE INVENTION

RNA interference (RNAi) describes a phenomenon in which the presence ofdouble-stranded RNA (dsRNA) having a sequence that is identical orhighly similar to a portion of a target gene results in the degradationof messenger RNA (mRNA) transcribed from that targeted gene (Sharp2001). Fjose et al. have proposed a mechanism for RNA interference[Fjose et al. RNA Interference: Mechanisms and Applications.Biotechnology Annual Review, Vol. 7, pp. 10-57 (2001)]. Initially adouble stranded RNA sequence (dsRNA) sequence is made available with onestrand that is identical or highly similar to a target gene andcomplementary to an mRNA produced from the transcription of the targetgene (the sense strand), and an antisense strand that is complementaryto the sense strand. Thus, the antisense sense strand has an identicalor highly similar sequence to a portion of the mRNA that results fromthe transcription of the target gene.

An RNAi nuclease then cleaves the dsRNA into short double strandedfragments whose lengths may vary from 18-25 nucleotides, and binds tothe mRNA produced from the transcription of the target gene. An RNAienzyme having helicase activity then catalyzes an exchange between theshort dsRNA and the mRNA so that the antisense strand of the dsRNAanneals to the mRNA, replaces the “antisense” strand of the dsRNA, andthe mRNA is cleaved at its ends. Consequently, the mRNA is destroyed,and translation of the mRNA molecule does not occur. Moreover, the sensestrand of the short dsRNA, which remains bound to the RNAi nuclease,serves as a template for production of a new antisense strand, forming anew dsRNA molecule for use in the destruction of another mRNA producedfrom the transcription of the target gene. Thus, RNAi demonstrates acatalytic activity. (Id.)

The ability to specifically knock-down expression of a target gene byRNAi has obvious benefits. For example, RNAi may be used to generateanimals that mimic true genetic “knockout” animals to study genefunction. In addition, RNAi may be useful in treating diseases ordisorders that arise from the abnormal expression of a particular geneor group of genes, or the expression of a gene having a particularmutation or polymorphism. For example, genes contributing to a cancerousstate (e.g., oncogenes) may be inhibited. In addition, viral genes maybe inhibited, as well as mutant genes causing genetic diseases such asmyotonic dystrophy, cystic fibrosis, Alzheimer's Disease, Parkinson'sDisease, etc. Inhibiting such genes as cyclooxygenase or cytokines mayalso have applications in treating inflammatory diseases such asarthritis.

Accordingly, what is needed is a vehicle that delivers heterologous RNAinto a cell in order to utilize interference RNA to modulate, andparticularly, to down-regulate the expression of a particular targetgene within a cell.

The citation of any reference herein should not be construed as anadmission that such reference is available as “Prior Art” to the instantapplication.

SUMMARY OF THE INVENTION

Provided herein is a useful and heretofore unknown retroviral viralvector that permits the delivery of heterologous RNA into a cell inorder to utilize RNA interference to modulate the expression of aparticular protein.

Broadly, the present invention extends to a retroviral vector forcarrying a target gene specific insert into a cell in order to modulatethe expression of a target gene. Such a retroviral vector of the presentinvention comprises a promoter, a polylinker region, and a target genespecific insert comprising double stranded RNA, which comprises a senseportion that is complementary to a portion of the antisense strand ofthe target gene, and an antisense portion that is complementary to thesense portion. Thus, the sense and antisense portions of the doublestranded RNA anneal, and the double stranded RNA folds back upon itself.

Numerous promoters have applications in a retroviral vector of thepresent invention. A particular example having applications in aretroviral vector of the present invention is the U6 promoter sequenceof:

(SEQ ID NO:7) ttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagtttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttgcctttatatatcttgtggaaaggacgaaacaccg.

Another promoter region having applications in a retroviral vector ofthe present invention is the H1 promoter, which has a nucleotidesequence of:

(SEQ ID NO:14) −1 ccctttctcaccagagtatgtcttgaatattctaagggtttaggtttctgtaaagtgcaaataccactaaagggtcttgtgtatcgctgtacgttta taa-100.

Likewise, numerous polylinker regions readily have applications in aretroviral vector of the present invention. Examples of polylinkerregions having applications in a retroviral vector of the presentinvention are

(a) aattc gactggcacagcctccagg ttcaagaga cctggaggctgtgccagtc ttttt ggaa a(SEQ ID NO:1) (b) aattc gctgggactcctttgcatg ttcaagagacatgcaaaggagtcccagc ttttt ggaa a; (SEQ ID NO:2) (c) gatccgactggcacagcctccagg ttcaagaga cctggaggctgtgccagtc ttttt ggaa a; (SEQ IDNO:3) (d) gatcc gctgggactcctttgcatg ttcaagaga catgcaaaggagtcccagc tttttggaa a (SEQ ID NO:4) (e) aattc gactccagtggtaatctac ttcaagagagtagattaccactggagtc ttttt ggaa a; (SEQ ID NO:5) and (f) gatccgactccagtggtaatctac ttcaagaga gtagattaccactggagtc ttttt ggaa a. (SEQ IDNO:6)

For a Lentivirus retroviral vector of the present invention, thepolylinker sequence can include an AgeI restriction site and an EcoRIrestriction site so that a target gene insert such as set forth belowcan be inserted:

AgeI +1      Loop     Term  EcoRI CCGGT G (20 more bases) TTCAAGAGA (21bases) TTTTT GGAA G     A C (20 more bases) AAGTTCTCT (21 bases) AAAAACCTT CTTAA (SEQ ID NOS: 15 and 16, respectively).

This insert contains AgeI and EcoRI restriction sites. The “20 or morebases” can be either the antisense or sense strand of the doublestranded nucleotide sequence of the target gene insert. Naturally the“21 bases” also can either the antisense or sense strand. However, it iscritical that both of these strands are complementary and anneal so thatthe double stranded RNA folds back upon itself. Moreover, the 9mer loopdescribed above is only an example. Other loops having other sizesreadily can be used in the present invention.

Furthermore, in a retroviral vector of the present invention, the lengthof the sense and antisense portions of the double stranded RNA in thetarget gene specific insert can vary. For example, the length of theseportions can be 19-30 nucleotides, in particular, 19-25 nucleotides, andmore particularly, 19-23 nucleotides, respectively.

Naturally, numerous genes can be the target gene for a retroviral vectorof the present invention. Particular examples of a target gene can be agene associated with a particular disease or disorder, e.g., an oncogenesuch as p53 or Mat8. A target gene can also be a gene associated with aneurodegenerative disease or disorder, such as, for example, a mutatedamyloid precursor protein or a presenilin gene that is associated withAlzheimer's disease. A target gene can also be a gene that encodes anion channel protein, a hormone, etc. Indeed, any gene for which it isdesirous to interrupt its expression has applications as a target genefor a retroviral vector of the present invention. In a particularexample described infra, the target gene is p38.

Numerous retroviruses have applications in a retroviral vector of thepresent invention. For example, a retroviral vector of the presentinvention may be constructed from a retrovirus such as HIV, MoMuLV(“murine Moloney leukaemia virus”), MSV (“murine Moloney sarcomavirus”), HaSV (“Harvey sarcoma virus”); SNV (“spleen necrosis virus”);RSV (“Rous sarcoma virus”), Friend virus, a murine stem cell virus(MSCV), a lentivirus, or even a defective retroviral vector such as thatdisclosed in WO95/02697, to name only a few. A particular retrovirushaving applications herein is a Murine Stem Cell Virus (MSCV). Anotherparticular retrovirus having applications herein is a modifiedLentivirus wherein (a) the endogenous CMV promoter has been removed; and(b) a REV element that binds to a REV response element (RRE) is insertedinto the virus.

Moreover, a retroviral vector of the present invention may furthercomprise a reporter gene, such as hrGFP, Blasti, Hygro, Puro, eGFP-Purofusion etc.

In another embodiment, the present invention extends to a cell infectedwith a retroviral vector of the present invention. Such an infection canoccur in vitro, in vivo, or ex vivo.

The present invention further extends to a modified Lentivirus vectorfor carrying double stranded RNA into a cell in order to modify theexpression of a target gene, wherein:

(a) the endogenous CMV promoter of the Lentivirus has been removed, themodified Lentivirus vector comprising:

-   -   (i) a REV element that binds to a REV response element (RRE) is        inserted;    -   (ii) a U6 promoter sequence of

(SEQ ID NO:7) ttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagtttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttgcctttatatatcttgtggaaaggacgaaacaccg; and

-   -   (iii) a polylinker region;    -   (iv) a target gene insert that comprises said double stranded        RNA,        wherein the double stranded RNA comprises a sense portion that        is complementary a portion of the antisense strand of the target        gene, and an antisense portion that is complementary to the        sense portion so that the sense portion and antisense portion        anneal, and the double stranded RNA folds back upon itself.

Numerous polylinker regions have applications in a modified Lentivirusvector of the present invention. Particular examples include, butcertainly are not limited to:

(a) aattc gactggcacagcctccagg ttcaagaga cctggaggctgtgccagtc ttttt ggaa a(SEQ ID NO:1) (b) aattc gctgggactcctttgcatg ttcaagagacatgcaaaggagtcccagc ttttt ggaa a; (SEQ ID NO:2) (c) gatccgactggcacagcctccagg ttcaagaga cctggaggctgtgccagtc ttttt ggaa a; (SEQ IDNO:3) (d) gatcc gctgggactcctttgcatg ttcaagaga catgcaaaggagtcccagc tttttggaa a (SEQ ID NO:4) (e) aattc gactccagtggtaatctac ttcaagagagtagattaccactggagtc ttttt ggaa a; (SEQ ID NO:5) and (f) gatccgactccagtggtaatctac ttcaagaga gtagattaccactggagtc ttttt ggaa a, (SEQ IDNO:6)to name only a few.

In a modified Lentivirus retroviral vector of the present invention, thepolylinker sequence can include an AgeI restriction site and an EcoRIrestriction site so that a target gene insert such as set forth belowcan be inserted:

AgeI +1                    Loop             Term     EcoRI CCGGT G (20more bases) TTCAAGAGA (21 bases) TTTTT GGAA G     A C (20 more bases)AAGTTCTCT (21 bases) AAAAA CCTT CTTAA

This insert contains AgeI and EcoRI restriction sites. The “20 or morebases” can be either the antisense or sense strand of the doublestranded nucleotide sequence of the target gene insert. Naturally the“21 bases” also can either the antisense or sense strand. However, it iscritical that both of these strands are complementary and anneal so thatthe double stranded RNA folds back upon itself. Moreover, the 9mer loopdescribed above is only an example. Other loops having other sizesreadily can be used in the present invention.

Furthermore, a modified Lentivirus of the present invention mayoptionally include a reporter gene, such as Blasti, hrGFP luciferase,etc.

Particular examples of a modified Lentivirus of the present inventiondescribed herein are

(a) pLenti-U6-Blasti, which comprises the nucleotide sequence of SEQ IDNO:8 (FIG. 1); and

(b) pLenti-U6-hrGFP, which comprises the nucleotide sequence of SEQ IDNO:9 (FIG. 2).

For a Lentivirus retroviral vector of the present invention, thepolylinker sequence can include an AgeI restriction site and an EcoRIrestriction site so that a target gene insert such as set forth belowcan be inserted:

AgeI +1       Loop     Term  EcoRI CCGGT G (20 more bases) TTCAAGAGA (21bases) TTTTT GGAA G     A C (20 more bases) AAGTTCTCT (21 bases) AAAAACCTT CTTAA (SEQ ID NOS: 15 and 16, respectively).

The present invention also extends to a Murine Stem Cell Virus (MSCV)vector for carrying double stranded RNA into a cell in order to modifythe expression of a target gene, comprising:

(a) a promoter; and

(b) a polylinker region,

(c) a target gene insert comprising the double stranded RNA, which inturn comprises a sense portion that is complementary a portion of theantisense strand of the target gene, and an antisense portion that iscomplementary to the sense portion so that the sense portion andantisense portion anneal, and the double stranded RNA folds back uponitself.

Various promoter sequences can be used in an MSCV retroviral vector ofthe present invention. A particular example of such a promoter sequenceis the U6 promoter sequence of

(SEQ ID NO:7) ttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagtttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttgcctttatatatcttgtggaaaggacgaaacaccg.

Another promoter having applications herein is the H1 promoter (SEQ IDNO: 14).

Furthermore, numerous polylinker regions have applications in a MSCVretroviral vector of the present invention. Particular examples include,but certainly are not limited to:

(a) aattc gactggcacagcctccagg ttcaagaga cctggaggctgtgccagtc ttttt ggaa a(SEQ ID NO:1) (b) aattc gctgggactcctttgcatg ttcaagagacatgcaaaggagtcccagc ttttt ggaa a; (SEQ ID NO:2) (c) gatccgactggcacagcctccagg ttcaagaga cctggaggctgtgccagtc ttttt ggaa a; (SEQ IDNO:3) (d) gatcc gctgggactcctttgcatg ttcaagaga catgcaaaggagtcccagc tttttggaa a (SEQ ID NO:4) (e) aattc gactccagtggtaatctac ttcaagagagtagattaccactggagtc ttttt ggaa a; (SEQ ID NO:5) and (f) gatccgactccagtggtaatctac ttcaagaga gtagattaccactggagtc ttttt ggaa a, (SEQ IDNO:6)

Optionally, a MSCV retroviral vector of the present invention can alsocomprise a reporter gene, such as Hygro, Puro, hrGFP, luciferase, oreGFP-Puro fusion.

Particular examples of MSCV retroviral vectors of the present inventioninclude

(a) MSCV-U6-Hygro, which comprises the nucleotide sequence of SEQ IDNO:10 (FIG. 3);

(b) MSCV-U6-Puro, which comprises the nucleotide sequence of SEQ IDNO:11 (FIG. 4); and

(c) MSCV-U6-hrGFP, which comprises the nucleotide sequence of SEQ IDNO:12 (FIG. 5), to name only a few.

Accordingly, it is an aspect of the present invention to provide aretroviral vector having a gene target insert that folds back uponitself to form a duplex, wherein one strand of the duplex is a sensestrand and the other strand of the duplex is an antisense strand. Whenthe retroviral vector is processed in the cell, the duplex is cleavedfrom the vector to form a short dsRNA for use as interfering RNA inmodulating the expression of the target gene.

It is another aspect of the present invention to provide a retroviralvector in which the sense strand of the target gene insert is identicalor highly similar to a target gene that is associated with a particulardisease or disorder. Such a retroviral vector may readily haveapplications in treating the disease or disorder associated with theexpression of the target gene.

It is another aspect of the present invention to provide a cell that hasbeen infected with a retroviral vector of the present invention. Suchinfection may occur in vitro, in vivo, or ex vivo. As a result of thisinfection, the expression of the target gene with the cell's genome ismodulated, and in particular, decreased.

It is still another aspect of the present invention to provide a methodfor modulating the expression of a target gene in cell using interferingRNA, wherein the cell is infected with a retroviral vector of thepresent invention comprising a target gene insert having a sense strandthat is identical or highly similar to the target gene.

These and other aspects of the present invention will be betterappreciated by reference to the following drawings and DetailedDescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Nucleotide sequence of modified Lentivirus vectorpLenti-U6-Blasti of the present invention containing the Blasti reportergene and the U6 promoter sequence. A target gene insert for modulating aparticular gene can readily be inserted into the polylinker of thisvector prior to infection of a cell with the vector.

FIG. 2: Nucleotide sequence of modified Lentivirus vectorpLenti-U6-hrGFP of the present invention containing the hrGFP reportergene and the U6 promoter sequence. A target gene insert for modulating aparticular gene can readily be inserted into the polylinker of thisvector prior to infection of a cell with the vector.

FIG. 3: Nucleotide sequence of MSCV vector MSCV-U6-Hygro of the presentinvention containing the Hygro reporter gene and the U6 promotersequence. A target gene insert for modulating a particular gene canreadily be inserted into the polylinker of this vector prior toinfection of a cell with the vector.

FIG. 4: Nucleotide sequence of MSCV vector MSCV-U6-Hygro of the presentinvention containing the Hygro reporter gene and the U6 promotersequence. A target gene insert for modulating a particular gene canreadily be inserted into the polylinker of this vector prior toinfection of a cell with the vector.

FIG. 5: Nucleotide sequence of MSCV vector MSCV-U6-hrGFP of the presentinvention containing the hrGFP reporter gene and the U6 promotersequence. A target gene insert for modulating a particular gene canreadily be inserted into the polylinker of this vector prior toinfection of a cell with the vector.

FIG. 6: a schematical view of a modified Lentivirus of the presentinvention that comprises a GFP reporter gene. (1) is the target geneinsert, i.e., the double stranded RNA that folds back upon itself.

FIG. 7: a western blot comparing the expression of p38 in a cellinfected with a modified Lentivirus of the present invention that lacksa target gene insert (a control) with the expression of p38 in a cellinfected with a modified Lentivirus of the present invention having atarget gene insert designed to be complementary to a portion of thecell's endogenous p38 gene. This blot clearly shows that the modifiedLentivirus of the present invention decreased the expression of p38relative to the expression in the control.

DETAILED DESCRIPTION OF THE INVENTION

As explained the above, the present invention broadly extends to auseful and heretofore unknown retroviral vector having applications indelivering interfering RNA into a cell to modulate, and moreparticularly to down-regulate the expression of a particular targetgene. Such a retroviral vector of the present invention comprises:

(a) a promoter,

(b) a polylinker region, and

(c) a target gene specific insert that comprises double stranded RNA,wherein the double stranded RNA comprises a sense portion that iscomplementary a portion of the antisense strand of the target gene, andan antisense portion that is complementary to the sense portion, so thatthe sense portion and antisense portion anneal, and the double strandedRNA folds back upon itself.

In a cell, the target gene specific insert that folds back upon itselfis processed into a short dsRNA duplex, of which the sense strand can beused as interfering RNA. This interfering RNA modulates, and moreparticularly, down-regulates the expression the target gene.Surprisingly and unexpectedly, a retroviral vector of the presentinvention that is used to infect a cell can down-regulate geneexpression of the target gene for the life of the cell, as opposed tothe mere insertion of naked siRNA into the cell, which has been found totypically down-regulate the expression of the target gene for only 5-6days.

Since a retroviral vector of the present invention is able todown-regulate the expression of the target gene, the retroviral vectormay have applications in treating a wide variety of diseases ordisorders related to the expression of a particular target, or relatedto the expression of a particular target gene that contains a mutationor polymorphism.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant nucleicacid molecule techniques within the skill of the art. Such techniquesare explained fully in the literature. See, e.g., Sambrook, Fritsch &Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989)Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

A “vector” is an agent, such as plasmid, phage, virus or cosmid, used totransmit genetic material to a cell or organism.

“Heterologous” nucleic acid molecule refers to a nucleic acid moleculenot naturally located in the cell, or in a chromosomal site of the cell.

A “nucleic acid molecule” or a “nucleotide sequence” can be usedinterchangeably, and refer to the phosphate ester polymeric form ofribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoesteranologs thereof, such as phosphorothioates and thioesters, in eithersingle stranded form, or a double-stranded helix. Double strandedDNA-DNA, DNA-RNA and RNA-RNA helices are possible.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., supra). The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. For preliminary screening for homologous nucleic acidmolecules, low stringency hybridization conditions, corresponding to aT_(m) of 55°, can be used, e.g., 5×SSC, 0.1% SDS, 0.25% milk, and noformamide; or 30% formamide, 5×SSC, 0.5% SDS). Moderate stringencyhybridization conditions correspond to a higher T_(m), e.g., 40%formamide, with 5× or 6×SSC. High stringency hybridization conditionscorrespond to the highest T_(m), e.g., 50% formamide, 5× or 6×SSC.Hybridization requires that the two nucleic acid molecules containcomplementary sequences, although depending on the stringency of thehybridization, mismatches between bases are possible. The appropriatestringency for hybridizing nucleic acid molecules depends on the lengthof the nucleic acid molecules and the degree of complementation,variables well known in the art. The greater the degree of similarity orhomology between two nucleotide sequences, the greater the value ofT_(m) for hybrids of nucleic acids having those sequences. The relativestability (corresponding to higher T_(m)) of nucleic acid hybridizationsdecreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybridsof greater than 100 nucleotides in length, equations for calculatingT_(m) have been derived (see Sambrook et al., supra, 9.50-0.51). Forhybridization with shorter nucleic acid molecules, i.e.,oligonucleotides, the position of mismatches becomes more important, andthe length of the oligonucleotide determines its specificity (seeSambrook et al., supra, 11.7-11.8). A minimum length for a hybridizablenucleic acid molecule is at least about 15 nucleotides; in particular atleast about 40 nucleotides; more particularly the length is at leastabout 30 nucleotides; more particularly at least about 60 nucleotides,and even more particularly at least 100 nucleotides.

Furthermore, as used herein, the term “standard hybridizationconditions” refers to a T_(m) of 55° C., and utilizes conditions as setforth above. In a preferred embodiment, the T_(m) is 60° C.; in a morepreferred embodiment, the T_(m) is 65° C.

A nucleic acid molecule “coding sequence” or “sense strand” is a nucleicacid molecule or portion thereof for eukaryotic genomic DNA molecules,which encodes a polypeptide or portion thereof with codons of thegenetic code in a correct reading frame. It is well known in the artthat the following codons can be used interchangeably to code for eachspecific amino acid:

Phenylalanine (Phe or F) UUU or UUC Leucine (Leu or L) UUA or UUG or CUUor CUC or CUA or CUG Isoleucine (Ile or I) AUU or AUC or AUA Methionine(Met or M) AUG Valine (Val or V) GUU or GUC of GUA or GUG Serine (Ser orS) UCU or UCC or UCA or UCG or AGU or AGC Proline (Pro or P) CCU or CCCor CCA or CCG Threonine (Thr or T) ACU or ACC or ACA or ACG Alanine (Alaor A) GCU or GCG or GCA or GCG Tyrosine (Tyr or Y) UAU or UAC Histidine(His or H) CAU or CAC Glutamine (Gln or Q) CAA or CAG Asparagine (Asn orN) AAU or AAC Lysine (Lys or K) AAA or AAG Aspartic Acid (Asp or D) GAUor GAC Glutamic Acid (Glu or E) GAA or GAG Cysteine (Cys or C) UGU orUGC Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG Glycine(Gly or G) GGU or GGC or GGA or GGG Tryptophan (Trp or W) UGGTermination codon UAA (ochre) or UAG (amber) or UGA (opal)

It should be understood that the codons specified above are for RNAsequences. The corresponding codons for DNA have a T substituted for U.

As used herein, the term “portion” with respect to a nucleotide sequencerefers to a part of said sequence having a length of at least 19contiguous nucleotides, but less than the entire nucleotide sequence.

A “promoter sequence” or “promoter” is a nucleic acid moleculeregulatory region capable of binding RNA polymerase in a cell andinitiating transcription of a downstream (3′ direction) coding sequence.Within the promoter sequence will be found a transcription initiationsite (conveniently defined for example, by mapping with nuclease SI), aswell as protein binding domains (consensus sequences) responsible forthe binding of RNA polymerase. A particular promoter sequence havingapplications in the present invention includes the U6 promoter sequence,which has the nucleotide sequence of:

(SEQ ID NO:7) ttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagtttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttgcctttatatatcttgtggaaaggacgaaacaccg.

Another example of promoter sequence having applications in a vector ofthe present invention is the H1 promoter (SEQ ID NO: 14).

As used herein, the terms “polylinker” or “polylinker region” can beused interchangeably, and refer to a nucleotide sequence that isinserted into a retroviral vector of the present invention and containsa plurality of restriction sites for particular restriction enzymes.Thus, using the particular restriction enzymes a nucleotide sequence canbe inserted into a vector of the present invention. Particular examplesof polylinkers having applications in a vector of the present inventioninclude, but certainly are not limited to:

(a) aattc gactggcacagcctccagg ttcaagaga cctggaggctgtgccagtc ttttt ggaa a(SEQ ID NO:1) (b) aattc gctgggactcctttgcatg ttcaagagacatgcaaaggagtcccagc ttttt ggaa a; (SEQ ID NO:2) (c) gatccgactggcacagcctccagg ttcaagaga cctggaggctgtgccagtc ttttt ggaa a; (SEQ IDNO:3) (d) gatcc gctgggactcctttgcatg ttcaagaga catgcaaaggagtcccagc tttttggaa a (SEQ ID NO:4) (e) aattc gactccagtggtaatctac ttcaagagagtagattaccactggagtc ttttt ggaa a; (SEQ ID NO:5) and (f) gatccgactccagtggtaatctac ttcaagaga gtagattaccactggagtc ttttt ggaa a. (SEQ IDNO:6)

In a particular embodiment of the present invention, e.g. a modifiedLentivirus retroviral vector, the polylinker sequence can include anAgeI restriction site and an EcoRI restriction site so that a targetgene insert such as set forth below can be inserted:

AgeI +1                    Loop             Term     EcoRI CCGGT G (20more bases) TTCAAGAGA (21 bases) TTTTT GGAA G     A C (20 more bases)AAGTTCTCT (21 bases) AAAAA CCTT CTTAA

This insert contains AgeI and EcoRI restriction sites. The “20 or morebases” can be either the antisense or sense strand of the doublestranded nucleotide sequence of the target gene insert. Naturally the“21 bases” also can either the antisense or sense strand. However, it iscritical that both of these strands are complementary and anneal so thatthe double stranded RNA folds back upon itself. Moreover, the 9mer loopdescribed above is only an example. Other loops having other sizesreadily can be used in the present invention.

As used herein, the term “infect” refers to the contamination of a cellwith a retroviral vector of the present invention, wherein the cellpossesses the gene target within its genome. Thus, infecting the cellwith a retroviral vector of the present invention with the proper targetgene insert will result in modulating the expression of the target genein the infected cell.

As used herein, the term “modulate” or “modulating” refers to alteringthe normal expression of a target gene in an infected cell. Inparticular, these terms refer to decreasing the amount expression of thetarget gene in the infected cell as compared to the amount of expressionof the target gene measured in the cell prior to infection with aretroviral vector of the present invention, or a decrease in the amountof expression of the target gene in the infected cell as compared to theexpression of the target gene in an uninfected control cell.

Retroviral Vectors

As explained above, the present invention extends to a retroviral vectorfor carrying a target gene specific insert into a cell in order tomodify the expression of a target gene, comprising:

(a) a promoter;

(b) a polylinker region;

(c) a target gene specific insert comprising double stranded RNA,wherein the double stranded RNA comprises a sense portion that iscomplementary a portion of the antisense strand of the target gene, andan antisense portion that is complementary to the sense portion, so thatthe sense portion and antisense portion anneal, and the double strandedRNA folds back upon itself.

Retroviruses are integrating viruses that infect dividing cells. Theretrovirus genome includes two LTRs, an encapsulation sequence and threecoding regions (gag, pol and env). Particular examples of retroviruseshaving applications herein, include, but certainly are not limited toretrovirus such as HIV, MoMuLV (“murine Moloney leukaemia virus” MSV(“murine Moloney sarcoma virus”), HaSV (“Harvey sarcoma virus”); SNV(“spleen necrosis virus”); RSV (“Rous sarcoma virus”), a Friend virus,and a defective retroviral vector such as one disclosed in WO95/02697,which hereby incorporated by reference herein in its entirety.

In general, in order to construct a recombinant retrovirus containing anucleic acid sequence, a plasmid is constructed which contains thenucleic acid sequence, which in the case of the present invention, is anRNA sequence that comprises a target gene specific insert as describedabove, and a polylinker region. Optionally, the RNA sequence can alsocomprise a nucleic acid that encodes a reporter protein. This constructis then used to transfect a packaging cell line, which cell line is ableto supply in trans the retroviral functions, which are deficient in theplasmid. In general, the packaging cell lines are thus able to expressthe gag, pol and env genes. Such packaging cell lines have beendescribed in the prior art, in particular the cell line PA317 (U.S. Pat.No. 4,861,719); the PsiCRIP cell line (WO90/02806) and the GP+envAm-12cell line (WO89/07150). After the construction, a retroviral vector ofthe present invention can be purified by standard techniques known tothose having ordinary skill in the arL A detailed description of theconstruction of a retroviral vector of the present invention is setforth infra.

A particular type of retrovirus having applications in a retroviralvector of the present invention is a modified Lentivirus, which is ableto infect post mitotic cells and/or non-dividing cells. Such types ofcells can be found in liver and muscle neurons. In a modified Lentivirusvector of the present invention, the endogenous CMV promoter of aLentivirus is removed, and a REV element is inserted into the virus.

Anther type of retrovirus having applications in a retroviral vector ofthe present invention is the MSCV virus.

Administration of a Retroviral Vector of the Invention Via Infection,Transfection or Transformation

The present invention further extends to a cell infected with aretroviral vector of the present invention, wherein the infected cellcontains the target gene within its genome. Hence, the infection of thecell with a retroviral vector of the present invention can modulate, andparticularly, decrease the expression of the target gene within thecell. Such infection can occur in vivo, in vitro, or ex vivo. Numeroustypes of cells can be infected with a retroviral vector of the presentinvention. For example, such a cell can be a prokaryotic or eukaryoticcell, e.g., bacterial cells such as E. coli, yeast cells or mammaliancells. Furthermore, such cells can be obtained from a biological samplesuch as, e.g., hair or skin, or body fluids, e.g., blood, saliva orsemen, etc. However, as explained above, it is important that the cellcontain the target gene within its genome.

Optionally, a cell can also be transformed or transfected with aretroviral vector of the present invention using routine laboratorytechniques, e.g., transfection, electroporation, microinjection,transduction, cell fusion, DEAE dextran, calcium phosphateprecipitation, lipofection (lysosome fusion), use of a gene gun, or aDNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem.267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624; Hartmut etal., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).

For a Lentivirus retroviral vector of the present invention, thepolylinker sequence can include an AgeI restriction site and an EcoRIrestriction site so that a target gene insert such as set forth belowcan be inserted:

AgeI +1       Loop     Term  EcoRI CCGGT G (20 more bases) TTCAAGAGA (21bases) TTTTT GGAA G     A C (20 more bases) AAGTTCTCT (21 bases) AAAAACCTT CTTAA (SEQ ID NOS: 15 and 16, respectively).

Pharmaceutical Compositions Containing a Retroviral Vector of theInvention

The present invention also extends to a pharmaceutical compositioncomprising a retroviral vector of the present invention and apharmaceutically acceptable carrier for the administration of aretroviral vector of the present invention. As used herein, the phrase“pharmaceutically acceptable” refers to molecular entities andcompositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like. Preferably, as used herein, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the compound is administered.Such pharmaceutical carriers can be sterile liquids, such as water andoils, including those of petroleum, animal, vegetable or syntheticorigin, such as peanut oil, soybean oil, mineral oil, sesame oil and thelike. Water or aqueous solution saline solutions and aqueous dextroseand glycerol solutions are preferably employed as carriers, particularlyfor injectable solutions. Suitable pharmaceutical carriers are describedin “Remington's Pharmaceutical Sciences” by E. W. Martin.

A pharmaceutical composition of the present invention may be foradministration for injection, or for oral, pulmonary, nasal or otherforms of administration, and may include diluents of various buffercontent (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength;additives such as detergents and solubilizing agents (e.g., Tween 80,Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodiummetabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) andbulking substances (e.g., lactose, mannitol); incorporation of thematerial into particulate preparations of polymeric compounds such aspolylactic acid, polyglycolic acid, etc. or into liposomes. Hylauronicacid may also be used. Such compositions may influence the physicalstate, stability, and rate of in vivo release. See, e.g., Remington'sPharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton,Pa. 18042) pages 1435-1712, which are herein incorporated by reference.The compositions may be prepared in liquid form, or may be in driedpowder, such as lyophilized form.

The present invention may be better understood by reference to thefollowing non-limiting Example, which is provided as exemplary of theinvention. The following Example is presented in order to more fullyillustrate the preferred embodiments of the invention. It should in noway be construed, however, as limiting the broad scope of the invention.

Example Cloning shRNA into Lentiviral Vector Called LUG

Initially, upper and lower strands of short hairpin nucleotide sequences(21 bases of sense strand, 9 bases of loop and 21 bases antisense) areobtained. They can be either produced or purchased from an oligo vender.The two strands are then in one well of a 96-well format.

Then, 50 pmol/ul of the strands are annealed, and 0.05 pmoles of theannealed strands are used to ligate. The oligos were such that the upperand lower strands were combined together in a single well of a 96-wellplate. All processes were carried out in a 96-well High-Throughputformat.

For the annealing reaction, 5 μl of the oligo mix was added to 45 μl ofthe annealing mix (40 μl water+5 μl NEB buffer 2). The mixture wasannealed by heating to 98° C. for 2 minutes in a thermocycler and thencooled on the bench top for at least 2 hours. The annealed oligos werethen diluted to 1:100 and 1 μl of the diluted annealed oligos, i.e., thedouble stranded DNA of the target gene insert, was ligated into thepolylinker of the retroviral vector backbone (which was pre-cut withAgeI and EcoRI). Ligations were carried out overnight at 4° C. 2 μl ofthe ligation was used to transform 40 μl of SURE cells (Stratagene) andthe transformations were plated on 12 well carbenicillin (50 μg/ml) gridplates. The following day, colonies were picked into SuperBroth-carbenicillin (50 μg/ml) and sent to HTP sequencing. The cultureswere then mini-prepped and sequenced. The sequences were analyzed andpositives were maxi-prepped to provide DNA for virus production.

Producing Modified Lentivirus in 293FT Cells:

Transfection Procedure:

One day prior to transfection, trypsinize and count the 293FT cells,plating them at 5×10 6 cells per 10 cm plate. Plate cells in 10 ml ofgrowth medium containing serum.

On the day of transfection, remove the culture medium from the 293FTcells and replace with 5 ml of growth medium containing serum (orOpti-MEM® I Medium containing serum). Antibiotics must not be included.

Prepare DNA-Lipofectamine {hacek over (Z)} 2000 complexes for eachtransfection sample by performing the following:

-   -   Dilute 9 μg of the optimized packaging mix and 3 μg of pLenti        expression plasmid DNA (12 μg total) in 1.5 ml of Opti-MEM® I        Medium without serum. Mix gently.    -   Mix Lipofectamine 2000 gently before use, then dilute 36 μl in        1.5 ml of Opti-MEM® I Medium without serum. Mix gently and        incubate for 5 minutes at room temperature.    -   After the 5-minute incubation, combine the diluted DNA with the        diluted Lipofectamine 2000. Mix gently.    -   Incubate for 20 minutes at room temperature to allow the        DNA-Lipofectamine 2000 complexes to form. The solution may        appear cloudy, but this will not impede the transfection.

4. Add the DNA-Lipofectamine 2000 complexes dropwise to each plate. Mixgently by rocking the plate back and forth. Incubate the cells overnightat 37° C. in a CO₂ incubator.

5. The next day, remove the medium containing the DNA-Lipofectamine 2000complexes and replace with complete culture medium (i.e. D-MEMcontaining 10% FBS, 2 mM L-glutamine, 0.1 mM MEM Non-Essential AminoAcids, and 1% penicmin/streptomycin).

A skilled artisan should note that expression of the VSV G glycoproteincauses 293FT cells to fuse, resulting in the appearance ofmultinucleated syncitia. This morphological change is normal and doesnot affect production of the lentivirus. It should also be noted that inpracticing the present invention, one is interacting with infectiousmaterials.

6. Harvest virus-containing supernatants 48-72 hours post-transfectionby removing medium to a 15 ml sterile, capped, conical tube. Minimaldifferences in viral yield are observed whether supernatants arecollected 48 or 72 hours post-transfection.

7. Centrifuge at 3000 rpm for 15 minutes at +4° C. to pellet celldebris.

8. Perform filtration step, if desired

9. Pipette viral supernatants into cryovials in 1 ml aliquots. Storeviral stocks at −80° C.

Producing MSCV in GP2-293 Cells:

Cell are maintained in a complete medium of DMEM supplemented with 10%FBS, 100 μg/ml streptomycin, 100 units/ml penicillin G.

Propagating Cells form Frozen Stocks:

1. Thaw vial in a 37° C. waterbath.

2. Transfer the cells to a tube containing 9 ml of pre-warmed completemedium.

3. Centrifuge in 1500 rpm for 5 minutes.

4. Remove supernatant.

5. Gently resuspend cells in 10 ml of complete medium and plate in a 10cm poly-D-lysine coated plate.

6. Incubate cells at 37° C. with 5% C02

poly-D-lysine coated plates can be used for the first week to promoteadherence after thawing. Subsequently, the cells may be cultured onregular plates.

Maintaining Packaging Cells

1. Aspirate medium, wash cells once with pre-warmed PBS.

2. Add 1 ml of trypsin-EDTA to the plate. Incubate for 30 sec-1 min.

3. Add 4 mls of complete medium to inhibit trypsin.

4. Resuspend the cells by pipetting up and down several times.

5. Transfer 1 ml of cells to a 10 cm plate containing 9 ml of completemedium.

The cells should be split 1:5 every 3 days when the cells are at 80%confluence. Moreover, cell should not be over trypsinized since as aresult, the cells tend to become clumpy and will not plate down well.Also, never let the cells get over confluent as this affects theirpackaging ability, and cells after Transfer/Passage #40 should not beused as their titers are compromised.Infection of GP2-293 Cells:

Day 1: (around 3 μm)

Plate 3 to 3.9×10⁶ cells per 10 cm plate (use poly-D-lysine coatedplates).

Day 2 (around 12 noon)

4 hours before the infection, re-feed the cells with 10 ml of freshmedium (minus antibiotics).

Infection Method 1 (Calcium Phosphate/HBS)

1. Use 12 μg of expression vector (PMK0.1) (DNA1) and 12 μg VSV-Gplasmid (DNA2) per transfection per 10 cm plate.

2. In a tube add:

DNA 1 12 μg

DNA 2 12 μg

A retroviral vector of the present invention can exist as a dsDNA vectorso that it can be propagated, such as in a plasmid. However, when it ispackaged, it is a retrovirus and infection leads to injection of thevirus nucleoprotein core (consisting mostly of gag-derived proteins, RNAvector, and the reverse transcriptase protein). Reverse transcriptaseconverts the retroviral vector of the present invention back into DNAand allows for stable integration into the genome of a cell infected.Furthermore, the DNA vector integrated (or transiently transfected)serves as the template for RNA polymerase III which binds to U6 promoterand transcribes shRNA from shDNA cloned into the vector. Above DNA1 isDNA vector (to deliver shRNA) and DNA2 is packaging plasmid.

Water

2M CaCl₂ 62 μl

Total Vol. 500 μl

3. In a separate tube, dispense 500 μl of 2×HBS.

4. Add the 2×BS dropwise to the DNA/CaCl₂ mixture whilst vortexing.

5. Incubate at room temperature for 20 minutes.

6. Vortex the DNA/CaCl₂ mixture gently.

7. Add the mixture to the packaging cells dropwise with a pipette.

8. Rock the plate back and forth to evenly distribute the solution.

9. Incubate the cells at 37° C. with 5% CO₂.

Transfection Method 2 (Lipofectamine 2000)

1. Use 6 μg of expression vector (pMK0.1) and 6 μg VSV-G plasmid pertransfection per 10 cm plate.

2. Add the DNA to a tube.

3. In a separate tube, pipette 72 μl of Lipofectamine 2000 into a 1.5 mlof serum-free medium (Optimem).

4. Mix gently. Incubate at room temperature for 5 minutes.

5. Add the Lipofecatamine/Optimem mixture to the DNA, mix gently.

6. Incubate at room temperature for 15 minutes.

7. Add the DNA/Lipo/Optimem mixture to the packaging cells dropwise witha pipette.

8. Rock the plate back and forth to evenly distribute the solution.

9. Incubate the cells at 37° C. with 5% CO₂.

Do not allow the transfection complex to sit on the cells for more than16 hours.

Day 3: (am)

1. Aspirate the medium.

2. Gently wash the cells once pre-warmed PBS

3. Add 5 mls of complete medium per 10 cm plate in order to concentratethe viral supernatant.

4. Incubate the cells at 37° C. with 5% CO₂.

Day 4: (am)

1. Harvest the medium, filter thru a 0.45 micron syringe filter.

2. Aliquot the virus, store at −80° C.

3. Re-feed the cells with 5 mls of complete medium.

This is the “24 hour viral supe” Harvesting the virus from the packagingcell line after 24 hours is 24 hour viral supernatant.

Day 5: (am)

1. Harvest the medium, filter thru a 0.45 micron syringe filter.

2. Aliquot the virus, store at −80° C.

3. Discard the plates.

This is the “48 hour viral supe” Harvesting the virus from the packagingcell line after 48 hours is 48 hour viral supernatant.

For Ecotropic Viruses, package in EcoPack Packaging Cell Line. For ANAmphotropic Virus, package in AmphoPack Packaging Cell line. For aPolytropic Virus, package in GP2-293 Packaging Cells (co-transfectvector with VSV-G expression plasmid)

Results

Using the procedures set forth above to produce a retroviral vector ofthe present invention, and then infecting cells having the target genein their genome with a retroviral vector of the present invention,expression of the target gene has been successfully decreased. In aparticular, the target gene insert for p38 was used, and the sequencefor this target insert is set forth below.

(SEQ ID NO:13) CCGGTGCAGGAGTTGAACAAGACAATACCTGATTGTCTTGTTCAGCTCCTGCTTTTTGGAAG.

FIG. 7 clearly shows that a modified Lentivirus of the present inventionhaving the target gene insert of double stranded RNA of SEQ ID NO:13,which was designed to interfere with expression of p38 in a cell,clearly decreased the expression of p38 in the cell relative to theexpression of p38 in a control cell.

The present invention is not to be limited in scope by the specificembodiments describe herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for description.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. A retroviral vector for carrying a target gene specific insert into acell in order to modify the expression of a target gene having a sensestrand and an antisense strand, comprising: (a) a U6 promoter having asequence of: (SEQ ID NO:7)ttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagtttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttgcctttatatatcttgtggaaaggacgaaacaccg; and

(b) a polylinker region comprising a nucleotide sequence of gatccgctgggactcctttgcatg ttcaagaga catgcaaaggagtcccagc ttttt ggaa a (SEQ IDNO:4) (c) a target gene specific insert comprising double stranded RNA,wherein said double stranded RNA comprises a sense portion that iscomplementary to a portion of the antisense strand of the target gene,and an antisense portion that is complementary to the sense portion, sothat the sense portion and antisense portion anneal, and the doublestranded RNA folds back upon itself.
 2. The retroviral vector of claim1, wherein the sense and antisense regions of the target gene specificinsert each comprise a length of 19-30 nucleotides.
 3. The retroviralvector of claim 2, wherein the sense and antisense regions of the targetgene specific insert each comprise a length of 19-25 nucleotides.
 4. Theretroviral vector of claim 3, wherein the sense and antisense regions ofthe target gene specific insert each comprise a length of 19-23nucleotides.
 5. A modified Lentivirus vector for carrying doublestranded RNA into a cell in order to modify the expression of a targetgene having a sense strand and an antisense strand, wherein: (a) theendogenous CMV promoter of the Lentivirus has been removed, saidmodified Lentivirus vector comprising: (i) a REV element that binds to aREV response element (RRE) is inserted; (ii) a U6 promoter sequence of(SEQ ID NO:7) ttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagtttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttgcctttatatatcttgtggaaaggacgaaacaccg; and

(b) a polylinker region comprising a nucleotide sequence of: gatccgctgggactcctttgcatg ttcaagaga catgcaaaggagtcccagc ttttt ggaa a (SEQ IDNO:4); wherein said double stranded RNA comprises a sense portion thatis complementary to a portion of the antisense strand of the targetgene, and an antisense portion that is complementary to the senseportion so that the sense portion and antisense portion anneal, and thedouble stranded RNA folds back upon itself.
 6. The modified Lentivirusvector of claim 5, further comprising a reporter gene.
 7. The modifiedLentivirus vector of claim 5, wherein said reporter gene is selectedfrom the group consisting of Blasti and hrGFP.
 8. The modifiedLentivirus vector of claim 7, wherein said vector is pLenti-U6-Blasti,which comprises the nucleotide sequence of SEQ ID NO:8.
 9. A modifiedlentivirus pLenti-U6-Blasti, comprising the nucleotide sequence of SEQID NO:8.
 10. A cell transformed or transfected with the modifiedlentivirus of claim 9.